Tipping the signaling scales

The strength of the signal produced by the T cell receptor (TCR) in response to self-antigen determines whether an immature thymocyte undergoes positive selection and matures into a T cell or negative selection and is eliminated to avoid autoreactivity. Both selection processes require the protein Themis1. By examining the effects of either loss or overexpression of Themis1 in thymocytes in mice, Zvezdova et al. determined that Themis1 enhances the activity of the guanine nucleotide exchange factor Vav1 and the stability of the TCR-associated adaptor protein Grb2, thus enabling TCR signaling. These data suggest that, although Themis1 also recruits a phosphatase to the TCR complex, the primary role for Themis1 is to enhance rather than inhibit TCR signaling to promote thymocyte development.

Abstract

The T cell signaling protein Themis1 is essential for the positive and negative selection of thymocytes in the thymus. Although the developmental defect that results from the loss of Themis1 suggests that it enhances T cell receptor (TCR) signaling, Themis1 also recruits Src homology 2 domain–containing phosphatase-1 (SHP-1) to the vicinity of TCR signaling complexes, suggesting that it has an inhibitory role in TCR signaling. We used TCR signaling reporter mice and quantitative proteomics to explore the role of Themis1 in developing T cells. We found that Themis1 acted mostly as a positive regulator of TCR signaling in vivo when receptors were activated by positively selecting ligands. Proteomic analysis of the Themis1 interactome identified SHP-1, the TCR-associated adaptor protein Grb2, and the guanine nucleotide exchange factor Vav1 as the principal interacting partners of Themis1 in isolated mouse thymocytes. Analysis of TCR signaling in Themis1-deficient and Themis1-overexpressing mouse thymocytes demonstrated that Themis1 promoted Vav1 activity both in vitro and in vivo. The reduced activity of Vav1 and the impaired T cell development in Themis1−/− mice were due in part to increased degradation of Grb2, which suggests that Themis1 is required to maintain the steady-state abundance of Grb2 in thymocytes. Together, these data suggest that Themis1 acts as a positive regulator of TCR signaling in developing T cells, and identify a mechanism by which Themis1 regulates thymic selection.

INTRODUCTION

Both the positive and negative selection of thymocytes are required to optimize T cell responses to foreign antigens and to prevent autoimmune reactions against self-antigens. The affinities of T cell antigen receptors (TCRs) for self-peptides bound to the major histocompatibility complex (pMHC) determine the fate of T cells during selection in the thymus. Negative selection eliminates T cells that strongly interact with self-pMHC molecules, which could otherwise result in autoimmunity, whereas positive selection promotes the survival and maturation of T cells with relatively low affinity for self-pMHC molecules, favoring the development of cells with greater self-reactivity within a permissible range constrained by the negative selection threshold (1). The recognition of pMHCs by the TCR stimulates multiple intracellular signaling events that are modulated quantitatively and qualitatively on the basis of the strength of TCR-pMHC interaction to selectively trigger selection processes and to promote genetic programs required for T cell maturation and egress from the thymus. Whereas the signaling pathways activated by TCR engagement have been identified, it remains unclear how the intensity of TCR stimulation precisely regulates the activation of downstream pathways such that two distinct outcomes [cell death (negative selection) or survival and continued maturation (positive selection)] can be elicited by engaging pMHC.

We and others identified the previously uncharacterized protein Themis1 as being required for the positive selection and maturation of T cells in the thymus (2–6). Themis1 lacks any discernable catalytic domain, but it contains two previously uncharacterized cysteine-based globular domains (named “CABIT”) of unknown structure and function (3). It also contains a bipartite nuclear localization signal (KR-X12-KRRPR) and a C-terminal proline-rich region (PRR) that matches a class II Src homology 3 (SH3) recognition motif. Themis1 is constitutively associated with the cytosolic adaptor protein Grb2 (growth factor receptor–bound protein 2) (5–7) and is recruited to the transmembrane adaptor protein LAT (linker of activated T cells) after TCR engagement, where it becomes rapidly phosphorylated by the protein tyrosine kinase Lck (8, 9).

Although Themis1 was identified as a component of the TCR signalosome, initial investigations failed to pinpoint a major alteration in TCR signaling in Themis1-deficient thymocytes; however, several experimental findings obtained from Themis1−/− mice are consistent with there being reduced TCR signal strength. The cell surface abundance of the signaling sensor CD5 is reduced in CD4+CD8loThemis1−/− thymocytes (5), whereas that of CD69 is decreased in the absence of Themis1 in preselected OT-I TCR transgenic CD4+CD8+ thymocytes [referred to as double-positive (DP) thymocytes] after stimulation through ovalbumin peptide–conjugated tetramers (2). In addition, negative selection and CD4+ T cell lineage commitment, which are dependent on intense or sustained TCR signals, respectively, are both impaired in Themis1−/− mice (2, 5). In agreement with these studies of rodent thymocytes, human Themis1 enhances extracellular signal–regulated kinase (ERK) phosphorylation and nuclear factor of activated T cells (NFAT)–activator protein 1 (AP1) luciferase promoter activity in Jurkat cells stimulated with TCR cross-linking antibodies (8). Further biochemical investigation to identify the molecular function of Themis1 has shown that it interacts with two other positive effectors of TCR signaling, phospholipase C-γ1 (PLC-γ1) (2) and Vav1, a guanine nucleotide exchange factor (GEF) Rho family guanosine triphosphatases (GTPases) (9), in thymocytes, but the importance of these interactions for TCR signaling and T cell development remains obscure.

In contrast to these initial studies, one recent report proposed that Themis1 sets the threshold between positive and negative selection by acting as an attenuator of TCR signaling during positive selection mediated by low-affinity antigens (10). It was shown in that study that the loss of Themis1 results in enhanced calcium (Ca2+) flux and increased phosphorylation of Lck, LAT, and ERK in preselected DP thymocytes that are stimulated by low-affinity pMHCs. Themis1 binds to the inhibitory protein tyrosine phosphatases SHP-1 (Src homology 2 domain–containing phosphatase-1) and SHP-2 in thymocytes (10) and Jurkat cells (11). The tyrosine phosphorylation of SHP-1 and its recruitment to LAT are reduced in Themis1−/− thymocytes, which suggests that Themis1 inhibits early signaling events by controlling the activity and localization of SHP-1. On the basis of these results, it was proposed that Themis1 functions to “digitalize” TCR signaling to prevent the generation of strong signals that induce negative selection in response to relatively low-affinity TCR–self-pMHC interactions. However, this model is inconsistent with results obtained in vivo that indicate that negative selection and CD4+ T cell lineage commitment are impaired in Themis1−/− mice. In addition, the proposed model is not fully supported by previous studies that showed that SHP-1 is not necessary for normal T cell development (12–15).

To gain further insight into the function of Themis1 during thymic selection, we analyzed the effect of Themis1 deficiency or overexpression on TCR signals elicited in vivo by positively selecting ligand interactions. Through experiments with TCR signaling reporter mice, we showed that Themis1 acted mostly as a positive regulator of TCR signaling during positive selection. Molecular analysis by quantitative mass spectrometry (MS) revealed that Themis1 interacted preferentially with Grb2, SHP-1, and Vav1, but to a lesser extent with SHP-2 and PLC-γ1. Analysis of TCR signaling events in cells treated with either low or high concentrations of TCR cross-linking complexes demonstrated that Themis1 promoted the phosphorylation and activity of Vav1 independently of the strength of TCR stimulation. Further biochemical analysis revealed that the reduced phosphorylation and activation of Vav1 in Themis1−/− thymocytes was due in part to the increased turnover of Grb2 in these cells, demonstrating a requirement for Themis1 in the maintenance of steady-state Grb2 protein abundance in thymocytes. Finally, we showed that positive selection was impaired in Grb2+/− mice and was rescued by the transgenic expression of Themis1, suggesting that the reduction in Grb2 abundance accounted for part of the developmental defect observed in Themis1−/− mice.

RESULTS

Signaling reporter mice identify a positive role for Themis1 in TCR signaling in vivo

Themis1 is a component of the TCR signaling machinery, but its precise function, and particularly whether it positively or negatively regulates TCR signaling responses, remains unclear. Some in vitro studies have proposed that Themis1 acts as a negative regulator of TCR signaling (10, 11); however, this is inconsistent with the developmental defect observed in Themis1−/− mice, which supports a positive role for Themis1 in TCR signaling (2, 3, 5, 9). To analyze the effect of Themis1 on the TCR signaling response to endogenous selecting ligands in developing thymocytes, we took advantage of the previously reported Nur77–green fluorescent protein (GFP) transgenic mouse model in which GFP abundance correlates with the intensity of TCR signals transmitted during positive or negative selection (16). We crossed Themis1−/− mice with Nur77-GFP transgenic mice expressing a fixed MHC class II–restricted αβ-TCR transgene (AND). GFP amounts were analyzed by flow cytometry ex vivo in preselection (TCRloCD69lo) CD4 and CD8 DP thymocytes, in postselection DP thymocytes at early (TCRloCD69hi) or late (TCRhiCD69hi) stages of positive selection, and in postselection CD4 single-positive (SP) thymocytes. As previously reported for mice on the wild-type C57BL/6 background, GFP abundance was low in preselection DP thymocytes and was increased in postselection DP thymocytes (Fig. 1A). GFP abundance was similar in preselection Themis1+/+ and Themis1−/− DP thymocytes, but was reduced in Themis1−/− DP thymocytes undergoing positive selection (Fig. 1A) and in Themis1−/− CD4 SP thymocytes (fig. S1A). To further analyze the influence of Themis1 on TCR signaling, we next crossed AND-Nur77-GFP transgenic mice with mice that overexpress Themis1 (Themis1-tg). In these mice, Themis1 abundance was increased by about threefold in DP thymocytes compared to that in DP thymocytes in nontransgenic wild-type controls (fig. S1B). GFP amounts were comparable in preselection Themis1+/+ and Themis1-tg DP thymocytes, but were increased in postselection Themis1-tg DP thymocytes (Fig. 1A) and in CD4 SP thymocytes (fig. S1A). Together, these data suggest that Themis1 has a positive role in the regulation of TCR signaling during positive selection.

(A) Contour plots represent the gating strategy to analyze GFP expression in preselection (TCRloCD69lo; red line in the GFP plot) and postselection (TCRloCD69hi or TCRintCD69hi; blue and green lines in the GFP plot, respectively) DP thymocytes from AND-Nur77-GFP transgenic mice. The abundance of the AND TCR was analyzed with anti-Vα11 antibodies. Histograms represent GFP expression in pre- and postselection DP thymocytes from AND-Nur77-GFP transgenic mice that either do or do not express the Bcl2 transgene (Bcl2-tg). Thymocytes from Themis1+/+ mice (Th+/+) were compared to those from Themis1−/− mice (Th−/−) or to Themis1 transgenic mice (Th-tg). Data are from one experiment and are representative of four independent experiments. (B) Left: Flow cytometric analysis of T cell development in Themis1+/+ and Themis1−/− mice that are either sufficient (Bim+/+) or deficient (Bim−/−) in the proapoptotic factor Bim. Contour plots represent either CD4 versus CD8 staining profiles of thymocytes (top) and splenocytes (bottom) or CD24 versus TCR staining profiles of CD4 SP thymocytes (middle). Numbers indicate the percentage of cells in the gated population. Right: Bar graphs present the mean percentages of CD4 and CD8 SP thymocytes (top), the mean ratio of mature CD24lo CD4 SP or CD24lo CD8 SP thymocytes to DP thymocytes (middle), and the mean percentages of splenic CD4+ and CD8+ T cells (bottom). Data are means ± SEM of three independent experiments containing one mouse of each genotype. *P < 0.05, **P < 0.01 by two-tailed, unpaired t test.

A previous report proposed that Themis1 acts as an attenuator of TCR signaling, such that in the absence of Themis1, positive selection signals, which promote the development of DP thymocytes to the SP stage, are converted to negative selection signals, leading to thymocyte apoptosis (10). On the basis of this model, the reduced amount of GFP that we observed in Themis1−/− mice might reflect the absence of “strongly signaled” thymocytes (which would have been eliminated by apoptosis) rather than an intrinsic decrease in TCR signal strength that occurred because of a positive function of Themis1 in TCR signaling. To exclude this possibility, we analyzed the effect of Themis1 deficiency on GFP protein abundance in transgenic mice that overexpressed the prosurvival factor B cell lymphoma 2 (Bcl2-tg), which blocks negative selection (17, 18). We observed that DP thymocytes from Bcl2-tg mice had greater amounts of GFP at early and late stages of positive selection than did equivalent cell populations from non–Bcl2-tg mice (Fig. 1A), which was suggestive of the overall rescue of thymocyte clones that had received a strong signal through the TCR. However, we found that the amount of GFP in Bcl2-tg–Themis1−/− DP thymocytes was reduced compared to that in Bcl2-tg–Themis1+/+ DP thymocytes (Fig. 1A). Together, these results suggest that the reduced TCR signaling observed in Themis1−/− thymocytes was not caused by the elimination of thymocytes that exhibited enhanced TCR signaling, but rather reflected an intrinsic defect in TCR signaling in DP thymocytes.

We previously showed that the transgenic expression of Bcl2 in Themis1−/− mice restores the development of CD8 SP thymocytes, but not CD4 SP thymocytes, which also suggests that the block in CD4+ T cell development in Themis1−/− mice did not result from an enhanced susceptibility of CD4 SP thymocytes to negative selection (5). It was proposed that disruption of Bim (a gene essential for negative selection) rescues CD4 SP development in Themis1−/− mice; however, a detailed analysis of thymocyte maturation and mature T cells was not performed in that study (10). To examine in greater detail whether the disruption of Bim could rescue SP thymocyte maturation in Themis1−/− mice, we recapitulated these experiments and analyzed the proportions of mature SP thymocytes (CD24lo) and peripheral T cells. We observed that, similar to the effect of Bcl2 overexpression, Bim disruption restored CD8 SP thymocyte development in Themis1−/− mice (Fig. 1B). However, the percentages and numbers of CD4 SP thymocytes and CD4+ splenic T cells remained reduced in Themis1−/−Bim−/− mice compared to those in Themis1+/+Bim−/− control mice (Fig. 1B and fig. S1C). The percentages of CD24lo CD4 SP thymocytes were also markedly reduced in Themis1−/−Bim−/− mice compared to those in Themis1+/+Bim−/− mice (Fig. 1B). These data suggest that the disruption of Bim does not rescue the block in CD4 SP T cell development in Themis1−/− mice, and support the notion that Themis1 acts as a positive regulator of TCR signaling that promotes positive selection.

Previous studies reported that Themis1 interacts with several positive (PLC-γ1, Grb2, SHP-2, and Vav1) and negative (SHP-1) regulators of TCR signaling, but the relative importance of these interactions remains unclear because some of these binding partners were identified in independent studies or within distinct cellular models through approaches that do not always enable global comparisons of protein-protein interactions. To determine which of these signaling proteins preferentially interacted with Themis1 during T cell development, and to investigate the mechanism by which Themis1 positively regulated TCR signaling, we performed an MS-based analysis of Themis1-containing signaling complexes in thymocytes. Themis1 was immunoprecipitated from wild-type or Themis1−/− thymocytes that were untreated or treated with pervanadate. Protein complexes were eluted with antigenic peptides, and the components of the different purified complexes were characterized by nanoflow liquid chromatography combined with tandem MS. To estimate the relative abundances of the different interacting partners in the immunoprecipitated samples, we used the intensity-based absolute quantification (iBAQ) metric, which corresponds to the sum of all of the peptide intensities divided by the number of theoretically observable tryptic peptides of a protein (for further details, see Materials and Methods).

On the basis of our selection criteria (see Materials and Methods), we identified 42 potential interacting partners of Themis1 in thymocytes (table S1). We decided to focus our analysis on the most abundant interactors (mean iBAQ > 20 × 105), which represented more likely direct binding partners of Themis1 or proteins that had a functional link with Themis1 (Fig. 2). Under these conditions, we found that Themis1 interacted mainly with distinct proteins in resting thymocytes (Fig. 2, A and B). Among these proteins was the cytosolic adaptor Grb2, the tyrosine phosphatase SHP-1 (also known as PTN6), and the Rho family GEF Vav1, which were previously reported to interact with Themis1 (5, 9, 10). In addition, we found that Themis1 constitutively interacted with the cytosolic adaptor protein GADS (also known as Grap2) and Lis1, a protein that has no reported function in T cells. Quantitative analysis of these interactions (with normalized iBAQ intensities) indicated that Grb2 was by far the most prominent binding partner of Themis1 in thymocytes (iBAQ = 980 × 105), whereas the interactions between Themis1 and SHP-1 (iBAQ = 30 × 105), Lis1 (iBAQ = 22 × 105), Grap2 (iBAQ = 15 × 105), and Vav1 (iBAQ = 10 × 105) were quantitatively similar (Fig. 2A). Treatment of thymocytes with the phosphatase inhibitor pervanadate increased either moderately (by about 2-fold for Grb2 and Lis1) or strongly (by about 10-fold for SHP-1, Vav1, and Grap2) the extent of the associations between these proteins and Themis1 (Fig. 2A). Treatment of thymocytes with pervanadate also induced the association of Themis1 with additional proteins, including the transmembrane adaptor proteins LAT and SIT1 (SHP-2–interacting transmembrane adaptor protein), the cytosolic adaptors Grap (GRB2-related adaptor protein) and SLP-76 (SH2 domain–containing leukocyte protein of 76 kD; also known as Lcp2), the E3 ubiquitin ligase c-Cbl, and the tyrosine phosphatase SHP-2 (also known as PTN11). Analysis of the iBAQ values indicated that the interaction between Themis1 and SHP-2 (iBAQ = 26 × 105) was not as preponderant as those between Themis1 and other effector proteins, such as SHP-1 (iBAQ = 378 × 105) and Vav1 (iBAQ = 97 × 105). Note that PLC-γ1, which was previously reported to interact with Themis1 in thymocytes, was detected in our current analysis with a statistically significant enrichment ratio in immunoprecipitated samples, but with only a low intensity value (iBAQ = 4 × 105), and thus was not selected in the list of major Themis1-interacting proteins (table S1).

Fig. 2Mass spectrometric analysis of the Themis1 interactome in thymocytes.

(A) Table representing the main interactors of Themis1 in resting (unstimulated) thymocytes and in pervanadate-treated (stimulated) thymocytes from C57BL/6 mice. Proteins were filtered and sorted on the basis of the enrichment ratio (m.i., mean intensity) and the mean iBAQ metric as described in Materials and Methods. The complete list of proteins identified by proteomic analysis of Themis1-immunopurified samples is provided in table S1. WT, wild type; KO, knockout. (B) Schematic of those proteins that preferentially interact with Themis1 in stimulated thymocytes. Layouts indicate the classification of these proteins according to their signaling function (purple layout: adaptor proteins; green layout: effector proteins; gray layout: unknown function in T cell signaling). Keys indicate protein classification according to molecular function (phosphatase, GEF, and E3 ubiquitin ligase). Data are representative of three independent experiments.

Themis1 enhances Vav1 activity in thymocytes

Our analysis of the Themis1 interactome identified SHP-1 and Vav1 as the two predominant effector proteins that bound to Themis1 in thymocytes. Vav1 is a known positive regulator of TCR signaling that promotes guanosine diphosphate (GDP) to guanosine triphosphate (GTP) exchange on Rho family GTPase proteins (that is, it has GEF activity) and may also serve as an adaptor protein to stabilize the recruitment of PLC-γ1 to the transmembrane adaptor protein LAT after TCR engagement (19). Knock-in mice expressing a GEF-deficient form of Vav1 exhibit a block in T cell development at the DP to SP transition, which is similar to the phenotype of Themis1−/− mice (20). We therefore suspected that the impaired T cell development observed in Themis1−/− mice might result, in part, from a defect in Vav1 GEF activity.

To first confirm the results obtained by our MS analysis, we used Western blotting to analyze the interaction between Themis1 and Vav1 in thymocytes stimulated with TCR cross-linking antibody complexes. Although Themis1 was coimmunoprecipitated with Vav1 from resting cells, the extent of the interaction between Themis1 and Vav1 was enhanced after TCR cross-linking (fig. S2A). Transfection of human embryonic kidney (HEK) 293T cells with complementary DNA encoding tagged versions of Themis1 and Vav1 showed that Vav1 coimmunoprecipitated with Themis1 only when cells were treated with pervanadate (fig. S2B). The coimmunoprecipitation of Vav1 with Themis1 was only mildly reduced when the PRR of Themis1, which is required for its interaction with Grb2, was deleted, indicating that Grb2 was not required for the interaction between Vav1 and Themis1 (fig. S2B).

We previously showed that the phosphorylation of Vav1 is impaired in Themis1−/− CD4 SP thymocytes (9); however, two studies suggested that Themis1 acts primarily as an attenuator of TCR signaling when thymocytes are stimulated by low-affinity antigens (10, 11). The phosphorylation of Vav1 and its downstream substrates was not examined in these studies. To recapitulate with cross-linking antibodies what was previously reported with antigenic peptides, we incubated thymocytes with either low (3 μg/ml) or high (30 μg/ml) concentrations of preformed complexes of anti-CD3 and anti-CD4 antibodies to induce either weak or strong TCR stimulation. Consistent with previous reports that used antigenic peptides, we found that ERK phosphorylation was increased in Themis1−/− thymocytes under weak stimulatory conditions, but was comparable to that in Themis1+/+ controls when strong stimuli were used to induce TCR signaling (Fig. 3A, top). The phosphorylation of SHP-1 was reduced in Themis1−/− thymocytes whether low or high concentrations of antibodies were used. The phosphorylation of Vav1 was decreased in Themis1−/− thymocytes when either weak or strong stimulation occurred (Fig. 3A). In comparison, the phosphorylation of Lck, ZAP-70 (ζ chain–associated protein kinase of 70 kD), LAT, and SLP-76, a cytosolic adaptor that associates with Vav1 (21), was similar in Themis1+/+ and Themis1−/− thymocytes regardless of the strength of stimulus (Fig. 3A and fig. S3).

(A) Left: Thymocytes from Themis1+/+ and Themis1−/− mice were stimulated with low (3 μg/ml) or high (30 μg/ml) concentrations of preformed anti-CD3 and anti-CD4 antibody complexes for the indicated times. Total cytoplasmic extracts of the cells were then analyzed by Western blotting with antibodies against phosphorylated forms of ERK, SHP-1, Vav1, SLP-76, and p38 MAPK. Right: Graphs show the relative abundances of the indicated phosphorylated proteins as determined by calculating the ratios of the intensities of the bands corresponding to the phosphorylated proteins to those corresponding to glyceraldehyde-3-phosphate dehydrogenase (GAPDH), the loading control. The y axes represent means ± SD of the relative values calculated after normalization to the highest value in the low-dose condition. Data are from four independent experiments each including one mouse of the indicated genotype. *P < 0.05, **P < 0.01 by two-tailed, unpaired t test. (B) Analysis of Vav1 phosphorylation by intracytoplasmic staining of thymocytes from AND-TCR transgenic mice that were either sufficient (Th+/+) or deficient (Th−/−) in Themis1. Histograms represent Vav1 phosphorylation on Tyr174 (Y174) in gated preselection (CD5lo) and postselection (CD5hi) DP thymocytes. Data are representative of three independent experiments each including one mouse of the indicated genotype. (C) Left: Thymocytes from Themis1+/+ and Themis1−/− mice were stimulated with high-dose anti-CD3 (α-CD3) and anti-CD4 (α-CD4) antibodies (30 μg/ml) for the indicated times. Cells were then lysed and subjected to Rac1-GTP pull-downs with a GST-PAK1-RBD fusion protein. Pull-downs and total cytoplasmic lysates were then analyzed by Western blotting (IB) with an anti-Rac1 antibody. Western blots are representative of three independent experiments each including one mouse of the indicated genotype. Right: Graphs show the ratios of Rac1-GTP to total Rac1. Data are representative of three independent experiments.

To demonstrate that Themis1 enhanced Vav1 activation during positive selection, we next analyzed the phosphorylation of Vav1 by intracytoplasmic staining in pre- and postselection DP thymocytes from Themis1−/− AND-TCR transgenic mice. We observed that postselection wild-type DP thymocytes had increased amounts of phosphorylated Vav1 compared to those of preselection wild-type DP thymocytes (Fig. 3B). Confirming the data obtained from experiments with TCR cross-linking antibodies, we found that Vav1 was less phosphorylated in Themis1−/− postselection DP thymocytes than in Themis1+/+ postselection DP thymocytes (Fig. 3B).

The phosphorylation of Tyr174 and Tyr160 of Vav1 relieves its catalytic Dbl homology domain from its autoinhibitory conformation and causes Vav1 to promote GDP to GTP exchange on the small GTPase Rac1 (22). The GTP-bound form of Rac1 then stimulates signaling that leads to the activation of the mitogen-activated protein kinase (MAPK) p38 (23, 24). To investigate the effect of Themis1 on signaling events downstream of Vav1, we analyzed the extent of phosphorylation of p38 and the abundance of Rac1-GTP generated after TCR cross-linking. The amount of phosphorylated p38 was reduced in Themis1−/− thymocytes compared to that in Themis1+/+ thymocytes when either low or high concentrations of antibody complexes were used (Fig. 3A). The amount of Rac1-GTP generated was also reduced in Themis1−/− thymocytes compared to that in Themis1+/+ control thymocytes when cells were stimulated with a high concentration of antibody complexes (Fig. 3C). Together, these data suggest that Themis1 enhances Vav1 activity in thymocytes.

The abundance of Grb2 protein is reduced in Themis1−/− thymocytes

We next investigated the mechanism by which Themis1 stimulates Vav1 activity in thymocytes. TCR cross-linking stimulates the translocation of Vav1 to the submembranous area and results in its recruitment to the transmembrane adaptor protein LAT where it becomes phosphorylated and activated by ZAP-70 (25). Because Themis1 interacts with both Vav1 and LAT after TCR engagement, we examined the possibility that Themis1 was important to enhance the recruitment of Vav1 to LAT. Similar amounts of Vav1 were coimmunoprecipitated with LAT from Themis1−/− and Themis1+/+ thymocytes at the earliest time point of stimulation with cross-linking antibodies (Fig. 4A); however, the recruitment of Vav1 to LAT was impaired in Themis1−/− thymocytes when the cells were stimulated for longer periods of time (Fig. 4A). In comparison, the recruitment to LAT of the enzyme PLC-γ1, which poorly interacts with Themis1 in thymocytes, appeared to be similar in Themis1+/+ and Themis1−/− thymocytes (Fig. 4A).

(A and B) Thymocytes from Themis1+/+ and Themis1−/− mice were stimulated with premixed high-dose anti-CD3 (α-CD3) and anti-CD4 (α-CD4) antibodies (30 μg/ml) for the indicated times. Samples were then subjected to immunoprecipitation (IP) with antibodies specific for LAT (A) or Vav1 (B) and then analyzed by Western blotting with antibodies specific for the indicated proteins. Western blots are from one experiment and are representative of three independent experiments. (B) Total cytoplasmic extracts (TCE) from each sample were analyzed by Western blotting with antibodies against Vav1 or Grb2. (C) Left: Total cytoplasmic extracts of thymocytes from Themis1+/+ (Th+/+), Themis1−/− (Th−/−), and Grb2+/− (Gr+/−) mice were analyzed by Western blotting with anti-Grb2 and anti-GAPDH antibodies. Right: Densitometric analysis of the ratio of Grb2 band intensities to GADPH band intensities normalized to the ratio in WT control thymocytes, which was set at 1. Data are means ± SD of four independent experiments each including one mouse of the indicated genotype. **P < 0.01 by two-tailed, unpaired t test. (D) Flow cytometric analysis of Grb2 abundance in DP, CD4 SP, and CD8 SP thymocyte subsets from Themis1+/+ (Th+/+), Themis1−/− (Th−/−), and Grb2+/− (Gr+/−) mice. Data are representative of three independent experiments. (E) Left: Thymocytes from Grb2+/+ and Grb2+/− mice were stimulated with low (3 μg/ml) or high (30 μg/ml) concentrations of preformed anti-CD3 and anti-CD4 antibody complexes for the indicated times. Total cytoplasmic extracts of the cells were then analyzed by Western blotting with antibody against pVav1. Right: Graphs show the relative abundance of pVav1, as determined from a ratio of the intensity of the pVav1 bands to those of the bands corresponding to the GAPDH loading control. Western blots and densitometry are from a single experiment and are representative of three independent experiments.

A possible explanation for this observation is that Themis1 directly interacts with Vav1 after its recruitment to LAT and that it contributes to the enrichment of Vav1 proteins in LAT signaling complexes. Alternatively, Themis1 might be required to stabilize the interaction between Vav1 and cytosolic adaptors, such as Grb2 and SLP-76, which mediate the binding of Vav1 to LAT (26–31).Therefore, we examined whether Themis1 regulated the interaction between Vav1 and these two adaptor proteins. Western blotting analysis showed that the amount of Grb2 that coimmunoprecipitated with Vav1 was decreased in Themis1−/− thymocytes compared to that in Themis1+/+ cells, regardless of whether the cells were stimulated with preformed antibody complexes (Fig. 4B). Similar amounts of phosphorylated SLP-76 were coimmunoprecipitated with Vav1 under the identical stimulation conditions, supporting a selective effect of Themis1 on the formation of Grb2-Vav1 signaling complexes (Fig. 4B). Furthermore, analysis of Themis1−/− thymocyte extracts revealed that the total cellular abundance of Grb2 was reduced relative to that in Themis1+/+ thymocytes (Fig. 4B, right panel). Quantification of Grb2 protein abundance in whole-cell lysates revealed that it was reduced by about twofold in Themis1−/− thymocytes and was comparable to the amount of Grb2 protein in Grb2+/− thymocytes (Fig. 4C). Further analysis by flow cytometry showed that Grb2 abundance was reduced similarly in DP and SP thymocytes from Themis1−/− mice (Fig. 4D), suggesting that the reduction in Grb2 abundance in Themis1−/− thymocytes relative to that in Themis1+/+ thymocytes was not a result of reduced numbers of SP thymocytes in Themis1−/− mice. The amounts of Grb2 that were coimmunoprecipitated with other known Grb2 binding partners, such as c-Cbl and Sos1, were also reduced in Themis1−/− thymocytes compared to those in wild-type controls (fig. S4). Moreover, and similar to what we previously observed for Vav1, the recruitment of Grb2 to LAT was normal at early time points after TCR stimulation but was progressively impaired in Themis1−/− thymocytes at later time points (Fig. 4A). To test whether the partial reduction in Grb2 abundance in Themis1−/− thymocytes accounted for the impaired activity of Vav1 in these cells, we compared the extent of Vav1 phosphorylation in Grb2+/+ and Grb2+/− thymocytes stimulated with low or high concentrations of preformed antibody complexes. Supporting this idea, Western blotting analysis showed that Vav1 phosphorylation was also reduced in Grb2+/− thymocytes under both stimulatory conditions (Fig. 4E).

Themis1−/− thymocytes exhibit increased turnover of Grb2 protein

To investigate the mechanisms leading to the reduction in Grb2 protein abundance in Themis1−/− thymocytes, we first quantified Grb2 mRNA by real-time polymerase chain reaction (RT-PCR) analysis. Grb2 mRNA amounts were similar in Themis1+/+ and Themis1−/− thymocytes, indicating that the reduction in Grb2 protein abundance in Themis1−/− thymocytes was likely a result of posttranscriptional effects (Fig. 5A). The direct interaction between Themis1 and Grb2 suggested the possibility that Themis1 might regulate Grb2 protein stability and turnover in thymocytes. To investigate this possibility, we incubated thymocytes from Themis1+/+ or Themis1−/− mice with cycloheximide for 18 hours to interrupt protein synthesis and then measured Grb2 protein abundance by intracellular staining and flow cytometry. Whereas the amount of Grb2 protein was reduced by about 10% in cycloheximide-treated Themis1+/+ thymocytes, it was reduced by 30% in Themis1−/− thymocytes (Fig. 5B), which suggests that Grb2 turnover was accelerated in Themis1−/− thymocytes. Next, we treated thymocytes with MG132 for 18 hours to inhibit proteasome-mediated protein degradation. Under these conditions, Grb2 abundance in Themis1−/− thymocytes increased from 60 to 80% of that in control Themis1+/+ thymocytes, demonstrating that blockade of proteasome-mediated protein degradation partially restored Grb2 abundance in Themis1−/− thymocytes (Fig. 5C). Because ubiquitylation plays a major role in targeting proteins for proteasome-mediated degradation, we evaluated the amount of ubiquitylated Grb2 in Themis1−/− thymocytes by Western blotting analysis. Under conditions in which similar amounts of Grb2 were immunoprecipitated from thymocyte lysates, the amount of ubiquitylated Grb2 was increased in Themis1−/− thymocytes compared to that in wild-type controls (Fig. 5D). Because c-Cbl was identified by our MS analysis as being among the preferential binding partners of Themis1 in thymocytes, we suspected that c-Cbl might regulate Grb2 degradation and that Themis1 might inhibit this process. However, we found that Grb2 protein amounts were unaffected in c-Cbl−/− thymocytes (fig. S5A). In addition, we found that the TCR-stimulated phosphorylation of c-Cbl was similar in Themis1+/+ and Themis1−/− thymocytes (fig. S5B).

(A) RT-PCR analysis of Grb2 mRNA abundance in total thymocytes from Themis1+/+, Themis1−/−, and Grb2+/− mice. Data are means ± SD of three independent experiments each including one mouse of the indicated genotype. (B) Thymocytes from Themis1+/+ (Th+/+) and Themis1−/− (Th−/−) mice were left untreated (black bars) or were treated (white bars) for 16 to 18 hours with cycloheximide (10 μg/ml). Grb2 protein abundance was then analyzed by flow cytometry after intracytoplasmic staining of the cells with anti-Grb2 antibodies. Bar graphs show relative mean fluorescence intensities (MFIs) of Grb2 in treated cells as a percentage of the MFI of Grb2 in untreated cells, which was set at 100%. Data are means ± SD of three mice of each genotype. (C) Thymocytes from Themis1+/+ (black bars) and Themis1−/− mice (white bars) were left untreated or were treated with 1 mM MG132 for 16 to 18 hours. Grb2 protein abundance was analyzed by flow cytometry after intracytoplasmic staining of cells with anti-Grb2 antibodies. Bar graphs show the relative MFIs of Grb2 in treated cells as a percentage of the MFI of Grb2 in Themis1+/+ thymocytes, which was set at 100%. Data are means ± SD of three independent experiments each including one mouse of the indicated genotype. (D) Thymocytes from Themis1+/+ and Themis1−/− mice were preincubated with MG132 and left untreated or treated with pervanadate (PV) for 5 min at 37°C. Grb2 was then immunoprecipitated from cellular extracts as described in Materials and Methods. Samples were analyzed by Western blotting with anti-ubiquitin and anti-Grb2 antibodies. Western blots are representative of three independent experiments each including one mouse of the indicated genotype. Data in (A) to (C) were analyzed by two-tailed unpaired t test. *P < 0.05, **P < 0.01.

The strength of TCR signaling was increased in vivo when Themis1 was overexpressed (Themis1-tg) in AND TCR transgenic thymocytes (Fig. 1A). We therefore examined the consequences of Themis1 overexpression on Vav1 phosphorylation and Grb2 protein abundance. The extent of Vav1 phosphorylation induced by low (Fig. 6A) or high (fig. S6A) concentrations of preformed anti-CD3 and anti-CD4 antibody complexes was increased in thymocytes in which Themis1 was overexpressed. Western blotting and flow cytometric analyses showed that Grb2 abundance was increased by about twofold in Themis1-tg thymocytes compared to that in Themis1+/+ controls (Fig. 6, B and C). In addition, the extent of the interaction between Grb2 and Vav1 and LAT was increased in Themis1-tg thymocytes compared to that in Themis1+/+ controls (fig. S6B).

(A) Left: Thymocytes from Themis1 WT (Themis1+/+) and Themis1 transgenic (Themis1-tg) mice were stimulated with preformed anti-CD3 and anti-CD4 antibody complexes (3 μg/ml) for the indicated times. Total cytoplasmic extracts were then analyzed by Western blotting with antibodies against pVav1 and pSLP76. GAPDH was used as a loading control. Right: Graphs show the relative abundances of the phosphorylated proteins as determined from a ratio of the intensity of the bands of phosphorylated proteins to those of GAPDH. The y axes represent means ± SD of the relative values calculated after normalization to the highest value in Themis1+/+ thymocytes. Data are from three independent experiments each including one mouse of the indicated genotype. *P < 0.05, **P < 0.01 by two-tailed, unpaired t test. Western blots are from one experiment and are representative of three independent experiments. (B) Flow cytometric analysis of Grb2 and Themis1 in total thymocytes from Themis1 transgenic mice (Tg) or littermate controls (WT). Data are representative of three independent experiments each including one mouse of the indicated genotype. (C) Left: Western blotting analysis of the relative amounts of Themis1, Grb2, and Vav1 in total thymocytes from Themis1 transgenic mice (Tg), WT littermate controls (T+/+), and Grb2+/− (Gr+/−) mice. Right: Bar graphs show the mean ratio ± SD of Grb2 band intensity values to Vav1 band intensity values, normalized to the ratio in T+/+ control thymocytes, which was set at 1. Data are means ± SD of three mice of each genotype. ‡P < 0.05 by two-tailed, unpaired t test. (D) Flow cytometric analysis of positive selection in AND Themis1 transgenic mice (Th-tg). Left: Contour plots represent the CD4 versus CD8 staining profiles of thymocytes from Themis1-tg mice and WT littermate controls (Th+/+) expressing the AND TCR. Histograms represent AND TCR surface staining with anti-Vα11 antibodies. Right: CD24 versus Vα11 staining profiles of CD4 SP thymocytes from Themis1-tg and littermate control mice expressing the AND TCR. Numbers indicate the percentage of cells in the gated population. Right: Bar graphs show the mean percentages of Vα11hi cells (top) and the mean ratio of mature CD4 SP (CD24lo) thymocytes to total DP thymocytes (bottom) in Themis1-tg and littermate control (WT) mice. Data are means ± SD of nine mice of each genotype. Five independent experiments were performed. *P < 0.05, **P < 0.01 by two-tailed, unpaired t test. Bottom: Cell surface staining of CD5 on gated DP and CD4 SP thymocyte subsets from Themis1-tg (Tg) and littermate control (Th+/+) mice. Data are representative of three independent experiments.

We next analyzed the consequences of Themis1 overexpression on T cell development. The percentages and numbers of CD4 and CD8 SP thymocytes were unaffected in Themis1-tg mice compared to that in littermate controls (fig. S7A). However, in Themis1-tg mice that also expressed the MHC class II–restricted AND transgenic TCR, the percentages of CD4 SP thymocytes and Vα11high thymocytes were increased relative to those in non–Themis1-tg controls (Fig. 6D). The percentage of mature CD24lo CD4 SP thymocytes was increased in AND–Themis1-tg mice, which is indicative of increased positive selection (Fig. 6D). In addition, the numbers of DP thymocytes, and, to a lesser extent, of CD4 SP thymocytes, were reduced in AND–TCR–Themis1-tg mice compared to those in non–Themis1-tg controls (fig. S7B), suggesting that the enhanced TCR signaling in Themis1-tg thymocytes may also partly convert positive selection signals into negative selection signals. The cell surface expression of CD5, a signaling marker that correlates with TCR signal intensity (32), was also increased on DP and SP thymocytes in AND–Themis1-tg mice (Fig. 6D), consistent with results obtained from Nur77-GFP transgenic mice (Fig. 1A).

Consistent with a previous report (33), the phosphorylation of Vav1 and p38 was reduced in TCR-stimulated Grb2+/− thymocytes compared to that in Grb2+/+ thymocytes (Fig. 4E). Because the overexpression of Themis1 in thymocytes resulted in increased Grb2 abundance and enhanced Vav1 phosphorylation in response to TCR engagement, we next examined whether overexpression of Themis1 could restore the amount of Grb2 and rescue the TCR signaling defects in Grb2+/− thymocytes. Western blotting analysis revealed that transgene-mediated overexpression of Themis1 increased the abundance of Grb2 in Grb2+/− thymocytes to amounts that were comparable to those in Grb2+/+ thymocytes (Fig. 7A). Moreover, phosphorylation of Vav1 and p38 in response to TCR engagement was restored when Themis1 was overexpressed in Grb2+/− thymocytes (Fig. 7B). Activation-induced phosphorylation of ERK was similar in Grb2+/+ and Grb2+/− thymocytes irrespective of whether the Themis1 transgene was expressed (Fig. 7B).

(A) Top: Total cytoplasmic extracts of thymocytes from Grb2+/+ and Grb2+/− mice that either expressed (+) or did not express (−) the Themis1 transgene (Themis1-tg) were analyzed by Western blotting with anti-Grb2 and anti-GAPDH antibodies. Bottom: Bar graphs show the mean ratio of Grb2 intensity values to GAPDH intensity values normalized to the ratio in WT control thymocytes, which was set at 1. Data are means ± SD of three independent experiments each including one mouse of the indicated genotype. (B) Thymocytes from Grb2+/+ and Grb2+/− mice that either expressed (+) or did not express (−) the Themis1 transgene (Themis1-tg) were stimulated with preformed anti-CD3 and anti-CD4 antibody complexes (30 μg/ml) for the indicated times. Total cytoplasmic extracts were analyzed by Western blotting with antibodies against the indicated phosphorylated proteins. Western blots are representative of three independent experiments. (C) Flow cytometric analysis of positive selection in AND-Grb2+/+ and in AND-Grb2+/− mice that did or did not express the Themis1 transgene (Themis1-tg). Contour plots represent CD4 versus CD8 staining profiles of thymocytes from mice of the indicated genotypes. Histograms represent the cell surface staining of the AND TCR on total thymocytes with anti-Vα11 antibodies. Numbers indicate the percentages of cells in the gated populations. Right: Bar graphs show the mean percentages of CD4 SP thymocytes (top) and Vα11high thymocytes (bottom) from mice of the indicated genotypes. Data are means ± SD of five mice (for Themis1+/+ and Grb2+/−) or three mice (for Themis1-tg and Themis1-tg/Grb2+/−). (D) Flow cytometric analysis of positive selection in AND ζ6Y/6Y and AND ζ6Y/6F mice that did or did not express the Themis1 transgene (Themis1-tg). Contour plots represent CD4 versus CD8 staining profiles of thymocytes from the indicated genotypes. Histograms represent AND TCR cell surface staining with anti-Vα11 antibodies on total thymocytes. Numbers indicate the percentages of cells in the gated populations. Right: Bar graphs show the mean percentages of CD4 SP thymocytes (top) and Vα11high thymocytes (bottom) from mice of the indicated genotypes. Data are means ± SD of three independent experiments each including one mouse of the indicated genotype. Data in (A), (C), and (D) were analyzed by two-tailed, unpaired t test. *P < 0.05, **P < 0.01.

We next examined whether Themis1 overexpression in Grb2+/− mice restored the defect in T cell development that occurs in these mice. Conditional deletion of Grb2 in thymocytes impairs T cell development at the DP to SP transition and induces a block in positive selection that resembles what is observed in Themis1−/− mice (34). A previous report demonstrated that negative selection is impaired in Grb2+/− mice but that positive selection is not affected (33). In that study, the authors used the MHC class I–restricted H-Y TCR transgenic and the MHC class II–restricted DO.11 TCR transgenic mice to evaluate positive selection. In contrast to these observations, we found that the proportions of CD4 SP thymocytes and Vα11high thymocytes were reduced by 40 to 50% in AND-Grb2+/− mice compared to that in AND-Grb2+/+ mice (Fig. 7C and fig. S8A). Positive selection was also impaired, although to a lesser extent, in OT-2–Grb2+/− mice (fig. S8B). Note that Themis1 overexpression corrected the defect in positive selection caused by Grb2 haploinsufficiency as demonstrated by the restoration of normal proportions of CD4 SP and either Vα11high (for AND) or Vα2high (for OT-2) T cells in the thymus (Fig. 7C and fig. S8, A and B).

To determine whether Themis1 overexpression restored T cell development in mice that exhibit a TCR signaling defect that is not linked to Grb2 signaling complexes, we crossed AND–Themis1-tg mice with knock-in mice that express a mutant CD3ζ chain protein that lacks signaling capability (ζ6F/6F). We previously reported that TCR signaling is attenuated in homozygous ζ6F/6F mice, which exhibit a block in positive selection at the DP stage. AND-ζ6Y/6F mice, which have one copy of wild-type ζ6Y and one copy of mutant ζ6F, displayed a less severe, but statistically significant, defect in positive selection (Fig. 7D and fig. S8C). The percentages of CD4 SP thymocytes and Vα11high thymocytes were reduced by about 70% in AND-ζ6Y/6F mice compared to those in AND-ζ6Y/6Y control mice (Fig. 7D). Consistent with a positive effect on TCR signaling, Themis1 overexpression in AND-Themis1 transgenic ζ6F/6Y mice increased the percentages of CD4 SP thymocytes and Vα11high thymocytes (Fig. 7D); however, Themis1 overexpression did not restore positive selection in ζ6F/6Y mice to the same extent as that observed in ζ6Y/6Y mice, suggesting that the positive effect of Themis1 on TCR signaling is restricted to Grb2 signaling complexes and does not compensate for the broad signaling defects caused by impaired TCR signaling potential (Fig. 7D and fig. S8C).

To further demonstrate that Themis1 enhances Grb2-mediated signaling during positive selection, we next used a reverse strategy and examined whether Themis1 hemideficiency (Themis1+/−) exacerbates the defect in TCR signaling and positive selection that is observed in Grb2+/− mice. The proportions of CD4 SP and Vα11hi thymocytes were decreased by 40 and 20%, respectively, in AND-Themis1+/− mice, an effect that was comparable to the defects observed in AND-Grb2+/− mice (Fig. 8A and fig. S8D). Note that positive selection was further impaired in AND-TCR transgenic mice that were hemizygous (+/−) for both Grb2 and Themis1, which displayed 70 and 60% decreases in the percentages of CD4 SP and Vα11high thymocytes, respectively, relative to those in AND Themis1+/+Grb2+/+ mice (Fig. 8A and fig. S8D). We also observed that the cell surface abundance of CD5 was reduced on Themis1+/−Grb2+/− thymocytes, but not on single heterozygote Themis1+/− thymocytes or Grb2+/− thymocytes, indicating that the combined reduction in Themis1 and Grb2 expression had a cooperative effect on TCR signaling and positive selection (Fig. 8B). Finally, hemideficiency in both Grb2 and Themis1 resulted in a further reduction in Grb2 cell surface abundance in thymocytes compared to that in thymocytes hemideficient in Grb2 or Themis1 alone (Fig. 8B).

(A) Flow cytometric analysis of positive selection in AND-Grb2+/+ and in AND-Grb2+/− mice that were either WT (Themis1+/+) or heterozygous (Themis1+/−) for Themis. Contour plots represent CD4 versus CD8 staining profiles of thymocytes from mice of the indicated genotypes. Histograms show the cell surface staining of the AND TCR on thymocytes with anti-Vα11 antibody. Numbers indicate the percentages of cells in the gated populations. Right: Bar graphs show the mean percentages of CD4 SP thymocytes (top) and Vα11high thymocytes (bottom) from mice of the indicated genotypes. Data are means ± SD of five mice (for Grb2+/+Themis1+/+ and Grb2+/−Themis1+/+) or three mice (for Grb2+/+Themis1+/− and Grb2+/−Themis1+/−). *P < 0.05 by two-tailed, unpaired t test. (B) Histograms show the cell surface staining of CD5 and the relative amounts of Grb2 in CD4 SP thymocytes from mice of the indicated genotypes. Data are representative of three independent experiments.

DISCUSSION

Initial studies reporting the identification and characterization of Themis1 included extensive analyses of T cell development in Themis1-deficient mice but failed to precisely identify the molecular function of this protein (2–6). Despite the absence of strong biochemical evidence, it was presumed that Themis1 likely functions as a positive regulator of TCR signaling because several defects in Themis1−/− mice, such as a marked impairment of both positive and negative selection, were consistent with a deficiency in TCR signaling (2–6). It was subsequently shown that Themis1 interacts with Vav1 and that Vav1 phosphorylation is reduced in Themis1−/− thymocytes, results that also implied a positive role for Themis1 in TCR signaling (9). Nevertheless, two reports suggested that Themis1 acts primarily, if not exclusively, as an attenuator of TCR signaling, in part through the recruitment of the phosphatase SHP-1 to LAT, and these studies further proposed that Themis1 is required to prevent the induction of strong TCR signals in response to engagement by low-affinity antigens (10, 11). Although this model can explain the block in positive selection in Themis1−/− mice, it does not fully explain the mechanism by which Themis1 also presumably regulates signaling during negative selection and CD4+ T cell lineage commitment. In addition, other studies have shown that positive selection is not converted to negative selection in the absence of SHP-1 (12–15), which suggests that Themis1 has additional effects on TCR signaling beyond the regulation of SHP-1.

To gain further insight into the molecular function of Themis1, we performed a quantitative proteomic analysis of the interactome of Themis1 in thymocytes. Analysis of this interactome showed that Themis1 preferentially interacted with proteins that function in TCR signaling, supporting a primary role for this protein in TCR signaling complexes. Among these proteins, we found that Themis1 interacted with several cytosolic adaptors, such as Grb2, GADS, SLP-76, and Grap, but that Grb2 was by far the most abundant protein in this interactome. This finding suggests that Themis1 might be preferentially recruited into TCR signaling complexes through Grb2. Confirming previous studies, we found that Themis1 interacted with the transmembrane adaptor protein LAT in pervanadate-treated thymocytes. Themis1 also interacts with SIT1, a transmembrane adaptor protein that inhibits TCR signaling through the phosphatase SHP-2 (35, 36). One possibility suggested by these data is that Themis1 is recruited to both LAT and SIT and that it promotes either positive or negative signals depending on the transmembrane adaptors to which it is recruited.

The quantitative analysis of the Themis1 interactome revealed that the phosphatase SHP-1, but not SHP-2, is a predominant interacting partner of Themis1 in thymocytes. Themis1 attenuates the phosphorylation of Lck, LAT, and ERK, mostly through its role in facilitating the recruitment of SHP-1 to TCR signaling complexes (10). This effect was most clearly demonstrated when the TCR was engaged by low-affinity, but not high-affinity, antigens, conditions that mimic positive selection. Confirming these results, we found that Themis1 enhanced the phosphorylation of SHP-1 and attenuated ERK phosphorylation when low, but not high, concentrations of TCR cross-linking antibody were used to stimulate thymocytes. However, we failed to detect any increased phosphorylation of more TCR-proximal signaling proteins, such as Lck, ZAP-70, and LAT, which suggests that stimulation with low concentrations of antibodies could not fully recapitulate what was previously observed when the TCR was engaged by tetramers bound to low-affinity antigens in the absence of costimulation. The molecular events that meditate ERK activation can be driven by hysteretic regulatory effects, and it is therefore assumed that minor changes in TCR-proximal signals can have a major effect on ERK activity (37, 38). It is therefore possible that TCR cross-linking with low concentrations of antibodies might not be sensitive enough to detect effects on early regulators of TCR signaling, but could still reveal the consequences on downstream proteins, such as ERK, that exhibit a digital mode of activation.

Our MS analysis also revealed that the Rho family GEF Vav1 constituted one of the predominant binding partners of Themis1 in thymocytes. Consistent with our previous results (9), we found that Themis1 enhanced Vav1 activity under both weak and strong stimulatory conditions. The phosphorylation of p38, which is dependent on Vav1 activity (23), was also reduced in Themis1−/− thymocytes compared to that in wild-type cells. This suggests that Themis1 has more than one function in TCR signaling and that the balance between the positive and negative effects of Themis1 on TCR signaling may be dependent on the extent (or intensity) of TCR engagement. Our findings suggest that Themis1 functions mostly as a positive regulator of TCR signaling with respect to its effect on Vav1 when selection processes are mediated by relatively high-affinity ligands. This interpretation would explain why negative selection, a process that is induced after the strong interaction of TCR with self-pMHCs, is impaired in Themis1−/− mice (2, 5). Our results further suggest that the strength of TCR signaling is reduced in Themis1−/− mice when positive selection is driven by a single transgene-encoded TCR (AND), a TCR that reportedly binds with relatively high affinity to self-pMHCs (1). Thus, the positive effect of Themis1 on the effector function of Vav1 might also be important to facilitate positive selection. Consistent with this idea, we found that the phosphorylation of Vav1 was reduced in vivo in postselection DP thymocytes from AND-Themis1−/− mice as compared to that in Themis1+/+ controls, and that the overexpression of Themis1 in Grb2+/− mice restored both Vav1 phosphorylation and positive selection driven by the AND TCR.

Our results demonstrated that the abundance of Grb2 was reduced in Themis1−/− thymocytes to an extent comparable to that in Grb2+/− thymocytes. Vav1 phosphorylation was also decreased in Grb2+/− thymocytes, which suggests that the reduced abundance of Grb2 in Themis1−/− thymocytes could account, at least in part, for the impaired phosphorylation of Vav1 in these cells in response to TCR engagement. Grb2 binds through its SH2 domain to phosphorylated tyrosine residues on LAT (28) and through its C-terminal SH3 domain to a PRR within Vav1 (26). Themis1 may therefore facilitate the recruitment of Vav1 to LAT and its subsequent phosphorylation by the tyrosine kinase ZAP-70. Accordingly, we showed that the recruitment of Vav1 to LAT was impaired in Themis1−/− thymocytes. A previous study of peripheral CD4+ T cells showed that the recruitment of Vav1 to TCR signaling complexes was mostly mediated by the binding of SLP-76 to the Vav1 SH2 domain through Tyr112 and Tyr126 of SLP-76 (39). In that study, deletion of the Vav1 SH2 domain prevented the recruitment of Vav1 to the immunological synapse and its subsequent phosphorylation. Here, we showed that loss of Themis1 did not affect the ability of Vav1 to interact with phosphorylated forms of SLP-76 after TCR engagement, which suggests that the impaired recruitment of Vav1 to LAT in Themis1−/− thymocytes resulted from the reduced Grb2 abundance in these cells, and that distinct mechanisms may control the recruitment of Vav1 into TCR signaling complexes in peripheral T cells and thymocytes.

Previous MS-based studies of the Grb2 interactome in distinct cell types have reported the interaction of Grb2 with proteins involved in ubiquitin-mediated degradation processes, such as c-Cbl, Cbl-b, TSG101, and SOCS1; however, whether these enzymes regulate Grb2 turnover has not been examined so far (40, 41). Because c-Cbl was identified by our MS analysis among preferential binding partners of Themis1 in thymocytes, we suspected that c-Cbl might regulate Grb2 degradation and that Themis1 might inhibit this process. However, we found that Grb2 protein amounts were unaffected in c-Cbl−/− thymocytes and that the interaction between c-Cbl and Grb2 was decreased rather than increased in Themis1−/− thymocytes compared to that in wild-type thymocytes. Although we cannot exclude the possibility that Cbl-b compensates for the loss of c-Cbl, this seems unlikely given that the abundance of Cbl-b in thymocytes is relatively low compared to that in peripheral T cells and that c-Cbl deficiency alone has a major effect on TCR abundance and signaling (42). In-depth analysis of the Themis1 interactome identified the deubiquitylating enzyme USP9X as a potential interactor of Themis1 in thymocytes (table S1). USP9X is part of the TCR signaling machinery and enhances TCR-proximal signaling events, such as the phosphorylation of LAT and Vav1 (43). One hypothesis, therefore, is that Themis1 prevents Grb2 degradation by interacting with a deubiquitylase that targets Grb2. Further experiments with USP9X-deficient T cells will be required to determine whether USP9X is involved in this process.

In contrast to previous studies with DO.11 TCR transgenic models, we found that positive selection driven by the AND or OT-2 TCR was partially attenuated in Grb2+/− mice. Because Grb2 abundance was similarly reduced in Themis1−/− and Grb2+/− thymocytes, this suggests that at least part of the defect in positive selection observed in Themis1−/− mice could be attributed to the reduced abundance of Grb2. However, the modest effect of Grb2 hemideficiency on T cell development in comparison to the marked defect observed in Themis1−/− mice implies that Themis1 regulates other T cell signaling processes independently of its effect on Grb2 protein stability. We showed here that the interaction between Themis1 and Vav1 did not require the binding of Grb2 to Themis1, which suggests that Themis1 might be important to recruit Vav1 directly into TCR signaling complexes. Another possibility is that Themis1 has a direct role in stimulating the enzymatic function of Vav1. Mice deficient in Vav1 GEF activity exhibit a phenotype similar to that of Themis1−/− mice, with a marked block at the DP to SP transition, which contrasts with the phenotype of Vav1−/− mice that exhibit an almost complete block at the β-selection checkpoint (20). Mice deficient in Vav1 GEF activity also exhibit a defect in Rac1-GTP production similar to what we observed in Themis1−/− mice. Furthermore, our MS analysis revealed that Themis1 interacted predominantly with the microtubule-associated protein Lis1, a direct binding partner of Rac1, which can stabilize the GTP-bound forms of small GTP-binding proteins (G proteins) in neurons (44). An interesting hypothesis is that Themis1 functions as part of a cooperative signaling complex that includes Grb2, Vav1, and Lis1 and controls the generation and the stability of active small G proteins, which in turn transmit signals that are essential for positive and negative selection.

In conclusion, our results identify a positive function for Themis1 in TCR signaling during T cell development and identify a primary role for Themis1 in promoting Grb2 stability and Vav1 effector function. These findings are concordant with a report demonstrating an epistatic effect of Themis1 and Vav1 genes on the suppressive function of regulatory T cells and the development of inflammatory bowel disease (45). In view of investigations that support a negative role for Themis1 in TCR signaling, our study suggests that Themis1 might have a more complex function during T cell development, perhaps transmitting either positive or negative signals depending on the affinity of the TCR for self-pMHCs, the contribution of co-receptor signaling, and the intensities of the elicited TCR-proximal signals. The identification of new Themis1 partners, such as Lis1, which is mostly known as a regulator of dynein anchoring to microtubules in neurons, suggests that Themis1 may have additional functions in T cells that are not directly related to its role in TCR-proximal signaling.

MATERIALS AND METHODS

Mice

Nur77-GFP reporter mice were provided by K. Hogquist (University of Minnesota, Minneapolis, MN). c-Cbl−/− mice were provided by J. Chiang (National Cancer Institute, Bethesda, MD). Themis1−/− (5), Themis1-tg (9), Grb2+/− (33), TCRζ6Y/6Y (46), and TCRζ6F/6F (46) mice were described previously. The AND and OT-2-TCR transgenic mice were obtained from Taconic Farms. The Bim−/− mice were obtained from the Jackson Laboratory. In some experiments, Themis1−/− and Themis1-tg mice expressing the AND TCR were crossed to Grb2+/− mice to generate either AND-TCR-Themis1+/−Grb2+/− or AND–TCR–Themis1-tg–Grb2+/− mice. Themis1−/− mice were also crossed with Bim−/− mice to generate Themis1−/−Bim−/− mice. Animal experiments were approved by the Animal Care and Use Committee of the Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health.

Antibodies and flow cytometry

Sources for antibodies and reagents used in this study include the following: anti-Grb2 (C-23), anti–PLC-γ1 (1249), anti-Vav1 (C-14), anti-ubiquitin (P4D1), and anti–c-Cbl (A9), which were from Santa Cruz Biotechnology; anti-LAT (11B.12), anti-Sos1, and anti-Rac1, which were from Millipore; anti-pVav1(Y174), which was obtained from Abcam; and anti-pERK (T202/Y204), anti-pp38 (T180/Y182), and anti–pSHP-1 (Y564), which were obtained from Cell Signaling Technology. Alexa Fluor 488–conjugated phalloidin was purchased from Life Technologies. Anti-Themis1 rabbit antibodies were previously described (5). Thymocytes were incubated for 16 hours with cycloheximide (20 μg/ml, Sigma) or 1 μM MG132 (EMD Biosciences). Biotin- and fluorochrome-conjugated antibodies against CD3ε, CD4, CD5, CD8α, CD24, CD25, CD44, and Vα11 were purchased from BD Biosciences. Single-cell suspensions from the thymus, spleen, or lymph nodes were incubated in phosphate-buffered saline, 0.5% bovine serum albumin, and 0.01% NaN3 containing the appropriate antibodies. Cell detection was performed on a FACSCalibur flow cytometer (BD Biosciences), and data analysis was performed with FlowJo software (Tree Star Inc.).

Thymocyte stimulation and immunoprecipitation

For MS analysis, 2 × 108 thymocytes from Themis1+/+ or Themis1−/− mice were left untreated or were treated with 100 nM pervanadate for 5 min at 37°C. The treatment was stopped on ice, and cells were immediately centrifuged and resuspended in 2 ml of ice-cold lysis buffer [10 mM tris-HCl (pH 7.4), 150 mM NaCl, 1% Triton, 2 mM Na3VO4, 5 mM NaF, 1 mM EDTA, and protease inhibitor cocktail tablet (Roche)] and incubated for 20 min on ice. Lysates were cleared by centrifugation at 18,000g for 15 min at 4°C, and Themis1 was subjected to immunoprecipitation from cleared lysates for 2 hours at 4°C with 30 μl of protein A–Sepharose resin coated with 12 μg of polyclonal rabbit anti-Themis1 antibodies. The resin was washed three times and incubated for 15 min at room temperature in 200 μl of elution buffer [50 mM tris-HCl (pH 7.4), 150 mM NaCl, and Themis1 antigenic peptide (200 μg/ml)]. Samples were further processed for proteomic analysis on a Q Exactive mass spectrometer (Thermo Scientific) as described in the Supplementary Materials. To analyze TCR signaling, thymocytes were collected and rested for 2 hours in RPMI medium at 37°C at a density of 5 × 106 cells/ml. Thymocytes were resuspended at a density of 107 cells per 50 μl and incubated at 37°C for 10 min before being stimulated. Antibody complexes were prepared at a 2× concentration before being used to stimulate cells at 37°C for 10 min with biotin-conjugated anti-CD3 and anti-CD4 antibodies mixed with equal concentrations of streptavidin. Thymocytes were stimulated with 50 μl of antibody complexes (6 or 60 μg/ml) for the times indicated in the figure legends. Cell lysates were prepared as described earlier. Further details about immunoprecipitations can be found in the Supplementary Materials.

Analysis of MS data

Raw MS data files were processed with MaxQuant software (version 1.5.0) for database search with the Andromeda search engine and for quantitative analysis (see the Supplementary Materials for details on the MaxQuant parameter settings and for the processing of quantitative data). Potential Themis1 interactors were selected on the basis of an enrichment ratio of >5 between immunopurified samples from Themis1+/+ and Themis1−/− cells and P < 0.05 (by Student’s t test) over triplicate biological experiments. Of the potential candidates, we retained as bona fide binding partners those that were identified in the Themis1+/+ immunopurified samples with strong mass spectrometric evidence (number of unique identified peptides > 2) and high abundance based on the intensity of the MS signal. The iBAQ metric, which corresponds to the sum of all of the peptide intensities divided by the number of observable peptides from a given protein (47), was used to estimate absolute protein abundance, and a minimal iBAQ threshold of 20 × 105 was thus applied to filter low-abundant proteins from the list of potential partners. Additionally, to define the Themis1 interactome, we also eliminated candidates that had strong intensity signals in the control Themis1−/− samples (mean iBAQ > 20 × 105), which were more likely to correspond to the nonspecific binding of abundant cellular proteins in the immunopurified samples. The remaining candidate interactors were sorted on the basis of their calculated iBAQ value in the Themis1+/+ immunopurified samples, which reflected their absolute abundance in the sample and the strength of their interaction with the Themis1 bait. The complete unfiltered list of proteins identified in the immunopurified samples is provided in table S1. The MS proteomics data were deposited to the ProteomeXchange consortium through the Proteomics Identifications (PRIDE) partner repository with the data set identifier PXD004072.

Rac1 activation assay

Thymocytes from Themis1+/+ and Themis1−/− mice were collected and rested for 2 hours in RPMI medium at 37°C at a density of 5 × 106 cells/ml. Cells were resuspended at 3 × 107 cells per 100 μl, preincubated at 37°C for 10 min, and then left untreated or treated with an equal volume of a premixed 2× cocktail of biotinylated anti-CD3 and anti-CD4 antibodies and streptavidin (100 μl) for the times indicated in the figure legends. Cells were lysed directly in excess lysis buffer (900 μl) for 20 min on ice. Lysates were cleared by centrifugation at 18,000g for 15 min at 4°C. Cleared lysates were subjected to pull-down with 10 μl of PAK1-GST agarose beads with rotation for 1 hour at 4°C. An aliquot of cleared lysates was saved for whole-cell lysate analysis. Beads were washed three times with ice-cold lysis buffer. Proteins were eluted from the beads by heating them to 70°C for 10 min in Laemmli buffer. Proteins were resolved by SDS–polyacrylamide gel electrophoresis (PAGE), subsequently transferred to polyvinylidene difluoride (PVDF) membranes, and analyzed by Western blotting to detect Rac1.

Western blotting analysis and band quantification

Proteins were resolved by SDS-PAGE and transferred to PVDF membranes according to standard protocols. Membranes were blocked with 5% milk in tris-buffered saline containing Tween for 1 hour at room temperature before being incubated with primary antibodies at 4°C overnight. After washing, membranes were incubated with secondary antibodies for 1 hour at room temperature. Subsequently, membranes were incubated with enhanced chemoluminescence solution for 5 min in the dark, and luminescence was captured with a Bio-Rad XRS+ imager. Images were analyzed, and band intensities were quantitated with Bio-Rad ImageLab software. Numbers from quantitated bands were normalized for loading differences with GAPDH as a control. In Fig. 3A, the intensity values at each time point were normalized to the highest intensity value obtained for wild-type thymocytes in the “low-dose” condition (which was set at 1) for each individual experiment.

RT-PCR analysis

For gene expression studies, total cellular RNA was isolated with a PicoPure RNA Isolation kit (Arcturus). RNA samples (100 ng each) were reverse-transcribed with the SuperScript First-Strand Synthesis system (Invitrogen) and were assayed by RT-PCR. Transcripts were quantified with a Roche LightCycler 480. Duplicates were run for each sample in a 96-well plate, and Actb served as the endogenous reference gene. The relative quantification method was used, with the ratio of the mRNA abundance of the gene of interest normalized to the abundance of Actb mRNA and with the average of the control thymocyte samples serving as the calibrator value. The specificity of the products was confirmed by melting curves and electrophoresis.

Statistical analysis

Statistical comparisons were performed with an F test to verify equal variance of the populations, which was followed by an unpaired two-tailed t test. The P values are indicated in the figure legends.

Acknowledgments: We thank K. Hogquist and J. Chiang for providing the Nur77-GFP transgenic mice and the cbl−/− mice, respectively; the Centre de Physiopathologie de Toulouse Purpan flow cytometry facility and the INSERM UMS006-CREFRE animal care facility; A. Saoudi for critical reading of the manuscript; and A. Mitra for the statistical analysis. Funding: This work was supported by INSERM and Sanofi (Avenir grant to R.L.); the Intramural Research Program of the Eunice Kennedy Shriver, National Institute of Child Health and Human Development; the Fondation pour la Recherche sur la Sclérose en Plaque (ARSEP); a Marie Curie International Reintegration Grant (R.L.); the French Ministry of Higher Education and Research (PhD fellowships for A.G., G.B., and J.A.); the Région Midi-Pyrénées; the Fonds Européens de Développement Régional, FEDER; the Toulouse métropole; and the French Ministry of Research with the Investissement d’Avenir Infrastructures Nationales en Biologie et Santé program (ProFI, Proteomics French Infrastructure project, ANR-10-INBS-08). Author contributions: E.Z. and J.L. performed flow cytometric analysis; J.M. and S.C. performed biochemistry; A.G., M.M., L.R., and J.F. performed experiments for MS; L.L. performed RT-PCR analysis; G.B. performed flow cytometric analysis of T cells in Bim−/−Themis1−/− mice; J.A. performed experiments for revision of the manuscript; M.M and A.G.d.P. performed analysis of MS data; A.G.d.P. and O.B.-S. supervised MS experiments; P.E.L. and R.L. designed the research and analyzed data; and R.L. wrote the manuscript. Competing interests: The authors declare that they have no competing interests. Data and materials availability: The MS proteomics data were deposited to the ProteomeXchange consortium through the PRIDE partner repository with the data set identifier PXD004072. The NIH requires materials transfer agreements for the distribution of the mouse lines generated for this study.